Inferring characteristics of bacterial swimming in biofilm matrix from time-lapse confocal laser scanning microscopy.

ABSTRACT

Biofilms are spatially organized communities of microorganisms embedded in a self-produced organic matrix, conferring to the population emerging properties such as an increased tolerance to the action of antimicrobials. It was shown that some bacilli were able to swim in the exogenous matrix of pathogenic biofilms and to counterbalance these properties. Swimming bacteria can deliver antimicrobial agents in situ, or potentiate the activity of antimicrobial by creating a transient vascularization network in the matrix. Hence, characterizing swimmer trajectories in the biofilm matrix is of particular interest to understand and optimize this new biocontrol strategy in particular, but also more generally to decipher ecological drivers of population spatial structure in natural biofilms ecosystems. In this study, a new methodology is developed to analyze time-lapse confocal laser scanning images to describe and compare the swimming trajectories of bacilli swimmers populations and their adaptations to the biofilm structure. The method is based on the inference of a kinetic model of swimmer populations including mechanistic interactions with the host biofilm. After validation on synthetic data, the methodology is implemented on images of three different species of motile bacillus species swimming in a Staphylococcus aureus biofilm. The fitted model allows to stratify the swimmer populations by their swimming behavior and provides insights into the mechanisms deployed by the micro-swimmers to adapt their swimming traits to the biofilm matrix.

Anyone who has ever cleaned a bathroom probably faced biofilms, the dark, slimy deposits that lurk around taps and pipes. These structures are created by bacteria which abandon their solitary lifestyle to work together as a community, secreting various substances that allow the cells to organise themselves in 3D and to better resist external aggression. Unwanted biofilms can impair industrial operations or endanger health, for example when they form inside medical equipment or water supplies. Removing these structures usually involves massive application of substances which can cause long-term damage to the environment. Recently, researchers have observed that a range of small rod-shaped bacteria ­ or 'bacilli' ­ can penetrate a harmful biofilm and dig transient tunnels in its 3D structure. These 'swimmers' can enhance the penetration of anti-microbial agents, or could even be modified to deliver these molecules right inside the biofilm. However, little is known about how the various types of bacilli, which have very different shapes and propelling systems, can navigate the complex environment that is a biofilm. This knowledge would be essential for scientists to select which swimmers could be the best to harness for industrial and medical applications. To investigate this question, Ravel et al. established a way to track how three species of bacilli swim inside a biofilm compared to in a simple fluid. A mathematical model was created which integrated several swimming behaviors such as speed adaptation and direction changes in response to the structure and density of the biofilm. This modelling was then fitted on microscopy images of the different species navigating the two types of environments. Different motion patterns for the three bacilli emerged, each showing different degrees of adapting to moving inside a biofilm. One species, in particular, was able to run straight in and out of this environment because it could adapt its speed to the biofilm density as well as randomly change direction. The new method developed by Ravel et al. can be redeployed to systematically study swimmer candidates in different types of biofilms. This would allow scientists to examine how various swimming characteristics impact how bacteria-killing chemicals can penetrate the altered biofilms. In addition, as the mathematical model can predict trajectories, it could be used in computational studies to examine which species of bacilli would be best suited in industrial settings.